C-linked glucuronide of N-(4-hydroxybenzyl) retinone, analogs thereof, and methods of using the same to inhibit neoplastic cell growth

ABSTRACT

Compounds of the formula: 
                         
are described, along with pharmaceutical compositions containing these compounds, and methods of using the compounds to prevent and to treat cancer in mammals, including humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This a continuation of co-pending application Ser. No. 12/357,737, filedJan. 22, 2009, which is a continuation of application Ser. No.11/416,907, filed May 3, 2006, now U.S. Pat. No. 7,528,114 which claimspriority to provisional application Ser. No. 60/677,503, filed May 3,2005, which is incorporated herein.

FEDERAL FUNDING STATEMENT

This invention was made with United States government support awarded bythe following agency: NIH CA049837. The United States government hascertain rights in this invention.

INCORPORATION BY REFERENCE

All of the references cited below are incorporated herein.

BACKGROUND

Retinol (compound 1) and its metabolites are involved in regulating manybiological processes including vision, cell differentiation, and growth.Besides being essential to normal cell function, the retinol metaboliteall-trans retinoic acid (RA, 2) also shows antiproliferative action incancer¹. At pharmacologically effective doses, however, RA causes severetoxicity. Therefore, development of retinoid analogs possessing a highertherapeutic index is needed. One of the most investigated syntheticretinoids is N-(4-hydroxyphenyl) retinamide (4-HPR; compound 3), whichhas been shown to be effective in numerous types of animal tumor modelsand has been evaluated in a phase III clinical trial.² A possiblebenefit was reported for the prevention of second breast malignancy inpremenopausal women with surgically removed stage I breast cancer orductal carcinoma in situ. Although 4-HPR is generally well tolerated, itresults in a decrease in plasma retinol levels^(3, 4) and concomitantdiminished dark adaptation. Dermatological disorders were reported in asubstantial number of subjects.⁵

Glucuronidation of drugs and natural products is a common metabolicpathway that usually facilitates excretion.⁶ An important metabolite of3 is 4-HPR-O-glucuronide (4-HPROG; compound 5) in which the phenolichydroxyl group is linked to the sugar. Subsequent to its discovery,compound 5 was synthesized and evaluated for bioactivity, and was shownto have excellent chemopreventative activity in a rat mammary tumormodel.⁷ However, it was not determined if the glucuronide 5, which wasshown to be hydrolyzed to compound 3 via β-glucuronidase,⁸ wasadvantageous due to improved bioavailability of 3 or had activity in itsown right as intact 5. To study this issue, an enzymatically stableglucuronide analog was synthesized by replacing the phenolic oxygen witha methylene group. The carbon-linked analog 4-HPR-C-glucuronide(4-HPRCG; compound 6) was shown to have excellent chemopreventative⁹ andchemotherapeutie₁₀ properties. Furthermore, much like 4-HPR, compounds 5and 6 show low affinity relative to RA for binding to the nuclearretinoic acid receptors (RAR), which mediate most of the actions ofnatural retinoids.⁹ Unlike compound 2, 4-HPR causes apoptosis innumerous cancer cell lines.¹¹ Thus the mode of action of these syntheticretinoids remains unclear.

While 4-HPR (compound 3) has been shown to be an effectivechemopreventative and therapeutic agent, some of its effects may beattributed to in vivo hydrolysis of the amide bond, liberating RA. Toinvestigate this possibility, an unhydrolyzable analog of 4-HPR,4-hydroxybenzyl retinone (4-HBR; compound 4) was synthesized. Bothcompounds 3 and 4 were shown to be equiactive chemotherapeutics in thedimethylbenz[a]anthracene (DMBA)-induced rat mammary tumormodel.^(12, 13) In vitamin A-deficient rats, compound 3, but notcompound 4, is hydrolyzed to liberate retinoic acid.¹³ Furthermore,4-HPR (3) but not the C-linked analog (4) induces CYP26B1 mRNAexpression in a RA-like manner in the lungs of vitamin A-deficient rats.Based on the positive chemotherapeutic and apoptotic-inducing activityof compound 4, it appears that hydrolysis of 4-HPR is not required forthe therapeutic effect of this retinoid, but rather, the liberation ofRA may contribute to its retinoid-based toxic side effects.

4-HPR has been shown to be 100 times less teratogenic than RA and thistoxicity may also be caused by the liberation of RA. With the effectiveantitumor agent 4-HPRCG (compound 6), amide bond hydrolysis may stilloccur in vivo, thus liberating retinoic acid by similar mechanisms asfor 4-HPR. Therefore, an unmet need exists for compounds that exhibitthe desirable anti-neoplastic activities of HPRCG, but which are notmetabolized in vivo to yield retinoic acid. The present invention isdirected to such compounds.

SUMMARY OF THE INVENTION

The invention is directed to compounds of Formula 1:

wherein X is CH₂; Y is C₁-C₆ alkylene; and R is selected from the groupconsisting of hydrogen, C₁-C₆ alkyl,

wherein R¹ is selected from the group consisting of H, OH, COOH, C₁-C₆alkyl, alkenyl, alkynyl, and C₁-C₆-hydroxyalkyl; R² is selected from thegroup consisting of H, OH and ═O; and salts thereof.

The “R” substituent can be in any stereochemical configuration (i.e., Dor L; R or S, etc.) as indicated by the wavy bonds depicted in the twostructures for “R” shown immediately above. Likewise, the9,13-dimethyl-substituted alkenylene chain, which is depicted in FormulaI in an all-trans conformation, may also include one or more cis doublebonds, at any position within the chain, but most notably at the 9 and13 positions. Thus, the present invention explicitly encompassescompounds of Formula I having any combination of cis or trans doublebonds within the alkenylene chain, such as (for example, and not by wayof limitation), the 9-cis isomer and the 13-cis isomer:

The invention is further directed to pharmaceutical compositions forpreventing and/or treating cancer in mammals. The compositions comprisean effective cancer cell growth-inhibiting amount of one or morecompounds as described herein (optionally in combination with apharmaceutically-suitable carrier).

The invention is further directed to a method of preventing and/ortreating cancer in mammals. The method comprises administering a cancercell growth-inhibiting amount of a compound or pharmaceuticalcomposition disclosed herein to a patient in need thereof, including ahuman patient.

The compounds are useful to prevent and to treat cancer in mammals,including humans. This utility is shown via the results of tumor growthinhibition assays using an accepted in vivo rat model of breast cancer.(See the Examples).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the effect of retinoid treatment ontime-course changes in DMBA-induced tumor volume in rats.

FIG. 2A is a bar graph depicting the effect of retinoid treatment onplasma triglyceride level. The reference “a” designated p<0.05 relativeto control, 4-HPR, and 4-HBRCG groups.

FIG. 2B is a bar graph depicting the effect of retinoid treatment onbone mineral content. Values are mean+SEM. The reference “a” designatedp<0.05 relative to the control group.

FIG. 3A is a graph depicting competition of retinoids for [³H]all-transRA binding to RARβ. ▪=4-HPR; □=4-HBRCGlucuronide; ♦=atRA;Δ=4-HBRCGlucose.

FIG. 3B is a graph depicting competition of retinoids for [³H]all-transRA binding to RARγ. ▪=4-HPR; □=4-HBRCGlucuronide; ♦=atRA;Δ=4-HBRCGlucose.

FIG. 4 is a bar graph depicting induction of CYP26A1 mRNA in the liverof retinoid-fed animals relative to the control group. Values aremean+SEM. Reference “a” designated p<0.05 relative to control and4-HBRCG groups; reference “b” designates p<0.05 relative to the RA-fedgroup.

FIG. 5 is a bar graph depicting reduction in live cell number in MCF-7cells treated with 4-HPRCG or 4-HBRCGlucose, relative to the vehiclecontrol group (V).

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions:

The following abbreviations and definitions are used throughout thespecification and claims. Terms not given an explicit definition hereinare to be interpreted according to their terms as understood in thefields of organic chemistry, pharmacology, and/or pharmaceuticalformulations.

Ac₂O=acetic anhydride.

AcOH=acetic acid.

atRA=all-trans retinoic acid.

9-BBN-H=9-borabicyclo[3.3.1]nonane.

BMC=bone mineral content.

Cp=cyclopentadienyl.

DMAP=4-dimethylaminopyridine.

DMBA=dimethylbenz[a]anthracene.

DMF=N,N-dimethylformamide.

DMX=dimethyl ketone, acetone.

dppf=1,1′-bis(diphenylphosphino)ferrocene.

EDTA=ethylenediaminetetraacetic acid.

Et₃N=triethylamine.

4-HBR=4-hydroxybenzyl retinone.

4-HBRCG=4-hydroxybenzyl retinone-C-glucuronide.

4-HBRCGlucuronide=4-hydroxybenzyl retinone-C-glucuronide.

4-HBRCGlucose=4-hydroxybenzyl retinone-C-glucose.

HPLC=high-performance liquid chromatography.

4-HPR=N-(4-hydroxyphenyl) retinamide.

4-HPRCG=N-(4-hydroxyphenyl) retinamide-C-glucuronide.

4-HPROG=N-(4-hydroxyphenyl) retinamide-O-glucuronide.

HRMS (ES)=high-resolution mass spectrometry, electrospray ionization.

IR=infrared.

LiHMDS=lithium hexamethyldisilazide.

MeOH=methanol.

MOM=methoxymethyl.

MOMCl=methoxymethylchloride.

mp=melting point.

mRNA=messenger ribonucleic acid.

NMR=nuclear magnetic resonance.

Ph=phenyl.

Pharmaceutically-suitable salts=Any acid- or base-addition salt whosecounter-ions are non-toxic to the patient in pharmaceutical doses of thesalts, so that the beneficial effects inherent in the free base or freeacid are not vitiated by side effects ascribable to the counter-ions. Ahost of pharmaceutically-suitable salts are well known in the art. Forbasic active ingredients, all acid addition salts are useful as sourcesof the free base form even if the particular salt, per se, is desiredonly as an intermediate product as, for example, when the salt is formedonly for purposes of purification, and identification, or when it isused as intermediate in preparing a pharmaceutically-suitable salt byion exchange procedures. Pharmaceutically-suitable salts include,without limitation, those derived from mineral acids and organic acids,explicitly including hydrohalides, e.g., hydrochlorides andhydrobromides, sulphates, phosphates, nitrates, sulphamates, acetates,citrates, lactates, tartrates, malonates, oxalates, salicylates,propionates, succinates, fumarates, maleates,methylene-bis-b-hydroxynaphthoates, gentisates, isothionates,di-p-toluoyltartrates, methane-sulphonates, ethanesulphonates,benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates,quinates, and the like. Base addition salts include those derived fromalkali or alkaline earth metal bases or conventional organic bases, suchas triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine,and the like.

Q-PCR=quantitative polymerase chain reaction.

RA=retinoic acid.

RAR=retinoic acid receptors.

RBP=retinol-binding protein.

rt=room temperature.

RXR=retinoid X receptor.

SEM=standard error of the means.

TBAF=tetrabutylammonium fluoride.

TBDMSCN=t-butyldimethylsilylcyanide.

TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy free radical.

THF=tetrahydrofuran.

TMS=tetramethylsilane.

Introduction:

To eliminate the possibility of enzymatic hydrolysis to release retinoicacid, the present invention is directed to a C-linked compound (4-HBRCG;compound 7) and analogs thereof. In 4-HBRCG, the amide bond of 4-HPRCGis replaced with a methylene group to yield the fully C-linkedderivative of 4-HPR-O-glucuronide, compound 7. The synthesis andtherapeutic evaluation of 4-HBRCG and analogs thereof are disclosed andclaimed herein.

Chemistry:

The recently reported improved synthetic route to 4-HPRCG employs aSuzuki coupling reaction between an exoanomeric methylene sugar and anaryl bromide.¹⁰ This methodology, originally developed by Johnson andcoworkers,^(14, 15) gives ready access to β-arylmethyl-C-glycosides.Using the same chemistries, with modifications noted herein, the keybenzyl bromide 14 was synthesized (see Scheme 1). Using a convergentapproach, an umpolung derivative of retinal was then alkylated with thebenzyl bromide to obtain the carbon skeleton of the final target,4-HBRCG (see Scheme 2).

Starting from readily available δ-D-gluconolactone (compound 8),hydroxyl protection using mild conditions with methoxymethylchloride(MOMCl) and diisopropylethylamine gives the protected lactone 9 (Scheme1). Olefination using Petasis reagent^(16, 17) gives the knownexoanomeric methylene sugar 10 in good yield.¹⁴ Hydroboration of theexocyclic olefin with 9-borabicyclo[3.3.1]nonane (9-BBN-H), followed bya Suzuki coupling with p-bromobenzyl alcohol, gives exclusively theβ-arylmethyl-C-glucoside, compound 11. Previous reports show thisreaction is stereoselective.^(10, 15, 18) The benzyl alcohol was easilyprotected to yield the methyl ether 12. To obtain the glucuronide, theMOM groups were cleaved in acid and the primary alcohol was selectivelyoxidized to the carboxylate using 2,2,6,6-tetramethyl-1-piperidinyloxyfree radical TEMPO.^(19, 20) Typically, TEMPO is used catalytically.However, these conditions resulted in nonselective oxidation, yieldingmixtures of benzylic ketones. Variations in the time, temperature, base,amount of TEMPO, amount of sodium hypochlorite, and order of additionwere attempted without any success in cleanly generating compound 13.From the inventors' past experience, other oxidatively sensitive,sugar-type molecules can undergo selective oxidations when excess TEMPOis used.²¹ When excess TEMPO, KBr, and NaClO are premixed in a NaHCO₃solution, deprotected 12 was added and selectively oxidized, efficientlyyielding the 6-position carboxylate. Methylation of the carboxylate,followed by acetylation of the remaining alcohols, gives the protectedC-aryl-glucuronide 13 in good yield over four steps. Benzylic methylethers can be displaced by bromide using hydrobromic acid^(22, 23) andwhen exposed to HBr in acetic acid, methyl ether 13 smoothly gave thekey benzyl bromide C-glucuronide intermediate 14. This surprisinglyfacile reaction yielded a very stable benzyl bromide, which was isolatedby crystallization.

Reagents and conditions for Scheme 1: (a) MOMCl, (i-Pr)₂NEt, Bu₄NI,CH₂Cl₂, 48 h, 83%; (b) Cp₂Ti(CH₃)₂, PhCH₃, 70° C., 18 h, 87%; (c) (i)9-BBN-H, THF, reflux, 6 h; (ii) PdCl₂(dppf), 3 M K₃PO₄, DMF,p-bromobenzyl alcohol, 18 h, 67%; (d) (i) NaH, THF, 1.5 h; (ii) CH₃I, 18h, 90%; (e) (i) 6 N HCl, MeOH, 18 h; (ii) TEMPO, NaClO, KBr, NaHCO₃, 0°C., 45 min; (iii) CH₃I, DMF, 20 h; (iv) Ac₂O, pyridine, DMAP, 18 h, 82%;(f) HBr, AcOH, 18 h, 86%.

The next step in this route was the key alkylation of electrophile 14with a retinal anion equivalent (see Scheme 2). The most suitableumpolung strategy for the chemically sensitive retinal is to employ theprotected cyanohydrin derivative,²⁴ and more particularly thesilylcyanohydrin²⁵ of retinal. The trimethyl-silylcyanohydrin of retinalwas first revealed and used in the synthesis of 4-HBR.^(12, 26) Retinal(compound 15, Scheme 2) exposed to t-butyldimethylsilylcyanide (TBDMSCN)with catalytic Et₃N gave chromatographically stable cyanohydrin 16. Inthe alkylation reaction, the TBDMS-cyanohydrin was deprotonated withLiHMDS and followed by addition of bromide 14. Subsequent chromatographyof the alkylated TBDMS-protected product 17 allowed for recovery of thevaluable unreacted bromide. Treatment of the alkylated product withfluoride unmasked the ketone to give the penultimate material 18. Usingmodel reactions, substantial efforts were undertaken to improve theyield of the alkylation reaction. Efforts included comparing TMS- andTBDMS-silylcyanohydrin reactivities, employing different bases, andchanging the stoichiometry, temperature, and time. Even though recoveryof unreacted bromide 14 was important, yields of 17 remained modest dueto sensitivities of the polyene retinoid reactant. Lastly, carefuldeprotection of the acetates and saponification of the methyl ester gavethe final target, 4-HBRCG (7). Using this synthetic route, more than 2grams of 4-HBRCG were produced to facilitate the animal studies and invitro assays disclosed herein.

Reagents and conditions for Scheme 2: (a) TBDSMCN, Et₃N, CH₂Cl₂, 20 h,78%; (b) (i) LiHMDS, THF, −78° C., 30 min; (ii) 14, THF, −78° C., 3 h,47%; (c) TBAF, THF, 1 h, 75%; (d) (i) K₂CO₃, MeOH, 4° C., 20 h; (ii)KOH, MeOH, 4° C., 20 h, 82%.

The other analogs disclosed herein can be fabricated by starting withthe appropriate initial reactants and then by following the samereaction routes and chemistries described immediately above and in theExamples. For example, the analogous 9-cis and 13-cis precursors can beobtained commercially from Sigma-Aldrich Chemicals (St. Louis, Mo.).

To fabricate the compounds wherein R² is hydroxy or a double-bondedoxygen (i.e., the 4-hydroxy and 4-oxo derivatives), the 4-hydroxy and4-oxo retinal reactants can be made using the methods described byCurley & Carson.⁴⁵

Biological Results and Discussion:

Preliminary evaluation of the mammary tumor chemotherapeutic activity of4-HBRCG (7) was conducted, and its toxicity profile was also assessed.As previously described,^(7, 12) tumor-bearing female rats (treated ca.50 days earlier with 7,12-dimethylbenz(a)anthracene [DMBA]) were fed thecontrol or retinoid-containing diets (RA (2), 4-HPR (3) or 4-HBRCG (7))at 2 mmole/kg diet for 22 days. As shown in Table 1, compound 7 is aseffective as 2 and 3 in reducing tumor volume (30-40% reduction),whereas control group tumor volumes increased nearly 200% by 22 days.FIG. 1 shows that the time course change in tumor volumes was similarfor all three treatment retinoids. Likewise, the data in Table 2 showsthat for compound 7, individual tumors in the group responded similarlyto the tumors in the other retinoid treated groups.

Of particular note, 4-HBRCG (7) showed evidence of greatly reducedtoxicity relative to both RA (2) and 4-HPR (3). As shown in Table 3,during the feeding period, only RA caused a significant reduction innormal body weight gain, a common sign of retinoid toxicity. Perhaps ofeven greater importance, 4-HBRCG caused a much smaller reduction thandid either 4-HPR or RA in plasma retinol levels. It is well known thatboth RA²⁷ and 4-HPR³ can reduce circulating levels of blood retinol. The4-HPR-induced reduction in plasma retinol has been show to produceimpaired dark adaptation, and is the single-most important factorlimiting the doses of 4-HPR that have been used in human clinicaltrials.²⁸ Because 4-HBRCG fed at 2 mmol/kg diet did not produce anysignificant reduction in blood retinol, whereas the same amount of 4-HPRdid, compound 7 can be administered at relatively higher doses comparedto 3 before the risk of night blindness is incurred.

While not being limited to any particular underlying biologicalmechanism, the reason that 4-HBRCG (7) shows no significant lowering ofblood retinol levels may be related to the manner in which it isdistributed in vivo (see Table 4). 4-HPR is known to reduce bloodretinol levels by competing for binding to the serum retinol-bindingprotein, RBP.^(3, 29) Interestingly, both 4-HPR and the related analog,4-HBR, show equivalent binding to RBP, yet 4-HBR does not lower bloodretinol levels.¹² It should be noted that 4-HBR circulates in the bloodat lower levels compared to 4-HPR when administered in equimolarquantities.¹² Thus, at least with respect to compounds 3 and 4, bloodretinol levels are inversely related to the concentration of 4-HPR thatis present in the blood. In the present invention, less 4-HBRCG waspresent in the plasma at the time the test animals were sacrificed ascompared to 4-HPR, suggesting that this decreased plasma concentrationmay account for the lesser effect of the glucuronide analog 7 oncirculating blood retinol levels.

TABLE 1 Effect of retinoid treatment on DMBA-induced rat mammary tumorvolume ^(a) Experimental Initial tumor Final tumor % group ^(b)volumes(cm³) volume (cm³) Change ^(c) Control 0.10 ± 0.05 0.29 ± 0.12+190 ^(d) Retinoic acid (2) 0.08 ± 0.03 0.05 ± 0.02  −38 ^(d) 4-HPR (3)0.12 ± 0.03 0.08 ± 0.02  −33 ^(d) 4-HBRCG (7) 0.12 ± 0.06 0.08 ± 0.05 −33 ^(d) ^(a) Value = mean ± SEM ^(b) Retinoid doses of 2 mmol/kg dietwere fed to animals for 22 days ^(c) Change from baseline ^(d) Denotesstatistical significance, p < 0.05

TABLE 2 Effect of retinoid treatment on individual tumors TotalExperimental number Complete Partial New No group of tumors regression^(a) regression ^(b) tumors effect Control 15 0 0 2 13  RA (2) 10 1 8 01 4-HPR (3) 19 1 15  0 3 4-HBRCG (7) 10 2 6 0 2 ^(a) Represents tumorsthat totally disappeared and could not be palpated ^(b) Representstumors that showed 25-75% decrease in volume

TABLE 3 Effect of dietary retinoid on body weight and plasma retinollevel Experimental % Weight group change ^(a) Plasma retinol ^(b)*Control +3 0.56 ± 0.10 Retinoic acid (2) +0.2 ^(d) 0.26 ± 0.03 ^(e)4-HPR (3) +3 0.21 ± 0.05 ^(e) 4-HBRCG (7) +3 0.40 ± 0.04 *Value = Mean +SEM ^(a) Relative to baseline body weight over 22 days ^(b)Concentration (μg/ml) at day 22 of treatment measured as in ref. 9 ^(c)Concentration (mg/ml) at day 22 of treatment measured as in ref. 24 ^(d)p < 0.05 for the trend in mean body weight gain relative to other groups^(e) p < 0.05 relative to control group

TABLE 4 Terminal plasma drug levels. Experimental group Plasma retinoidconcentration ^(a) Retinoic acid (2) 0.30 ± 0.10 4-HPR (3) 0.67 ± 0.074-HBRCG (7) 0.10 ± 0.01 ^(a) Concentration (μg/ml) ± SEM at day 22 oftreatment

As shown in FIG. 2A, treatment with RA dramatically increased serumtriglyceride concentration, whereas 4-HBRCG did not cause thisundesirable effect. An increase in serum triglyceride is a well knownside effect of oral RA administration,^(30, 31) and is mediated bybinding to the RAR family.³² As borne out in human trials, 4-HPR isclearly less potent than RA in producing this side effect.⁵ It ispossible that hydrolysis of 4-HPR may have accounted for the small butinsignificant increase upon feeding of this retinoid in the presentwork, whereas the non-hydrolyzable 4-HBRCG showed no propensity toincrease serum triglyceride levels.

Skeletal abnormalities are another adverse effect of high-dose retinoidtherapy.³³⁻³⁵ In order to determine the extent of any similar effect for4-HPR and the analog 7, the bone mineral content (BMC) of the femur ofanimals was measured at the end of the feeding study. As expected, RAproduced a significant reduction in femur BMC as compared to controlanimals (see FIG. 2B), whereas the groups receiving either 3 or 7 showedno such effect. In an early clinical study of women with early breastcancer receiving 4-HPR, a trend toward an increase in bone resorptionmarkers was noted.³⁶ Although not significant, this suggests that a4-HPR analog such as 4-HBRCG that cannot liberate RA might beadvantageous in minimizing bone risk.

As with 4-HPR and other related analogs (5 and 6), 4-HBRCG binds poorlyto the retinoic acid receptors β and γ (RARβ and RARγ) (see FIGS. 3A and3B). 4-HPR was nearly 3000 times less potent than all-trans retinoicacid (atRA) in competing for [³H]atRA binding to the RARβ, and 2500times less able to compete for binding to the RARγ.^(9, 37) In thepresent work, 4-HBRCG also showed only weak RAR binding (300 times and1400 times less potent than atRA in binding to RARβ and RARγ,respectively). 4-HBRCG at concentrations up to 10^(−4.5) M showedsimilar poor binding to the RARα (data not shown). Furthermore, 4-HBRCGat concentrations up to 10^(−4.5) M showed almost no competition for[³H]9-cis RA binding to the RXR (data not shown). Similar to 4-HBRCG,the 4-HBRCGlucose analog (23) also binds poorly to the RARβ and RARγ(see FIGS. 3A and 3B, respectively), and RARα and RXRγ (data not shown).

To evaluate the ability of retinoids to activate RAR-mediated genetranscription in vivo, the induction of CYP26 was measured at sacrificein liver and lung. RA was highly effective in inducing CYP26A1 mRNA inthe liver (67-fold above control; see FIG. 4) and CYP26B1 in the lung(46-fold above control; data not shown). 4-HPR also showed significantactivity in inducing the CYP26 mRNAs in liver and lung (37- and 20-foldfor CYP26A1 and CYP26B1, respectively, compared to control), whereas4-HBRCG did not induce these cytochrome p450 mRNAs. RA has been shown toinduce the CYP26A1 mRNA via binding to RARs and to direct interaction ofthe liganded RAR/RXR heterodimer with a retinoic acid response elementin the promoter region of this RA-responsive gene.^(38, 39) It has alsobeen shown previously that RA, and to a lesser extent 4-HPR, induces theexpression of CYP26B1 mRNA in lungs of vitamin A-deficient rats.¹³ Thefact that neither compound 3 nor compound 7 show particularly strongbinding to the RARs, coupled with the finding that 4-HBRCG actuallyshows enhanced affinity compared to 4-HPR for the RARs yet does notinduce gene expression, argues against a direct interaction of 4-HPRwith the receptor as a mechanism to explain its ability to induce thesemRNAs. Rather, hydrolysis of 4-HPR to atRA may account for thisinduction. It has been shown previously that 4-HPR given orally tovitamin A-deficient rats generates RA in plasma that is detectable byHPLC.¹³ The lack of induction of RAR-mediated gene transcription by4-HBRCG in vivo thus supports the conclusion that direct binding of 7 toRARs does not occur at the retinoid levels fed in the present work, andfurther indicates that RA-mediated toxicities should be less of aproblem with the present compounds as compared to 4-HPR.

Although the toxicity of 4-HPR has been reported to be reduced comparedto RA, the spectrum of toxicities encountered are similar.⁴⁰ Thus,4-HBRCG shows significant improvement in the therapeutic window comparedto the natural hormone, RA, for all measures of toxicity studied here(weight loss, elevation of serum triglyceride, reduction in bone mineralcontent, reduced blood retinol, and induction of RAR-mediated genetranscription) and for the latter two measures of toxicity when comparedto 4-HPR.

Inhibition of MCF-7 human breast cancer cell growth over an 8-day periodwas used as a means to assess the activity of 4-HBRCG and the analog4-HBRCGlucose. Both compounds produced a reduction in the number of livecells at 10⁻⁵ M. See FIG. 5. However, in the case of the 4-HBRCGlucoseanalog, the majority of the MCF-7 cells were dead at the end of theassay. This result strongly suggests that the glucose analog might be aneven more potent compound than 4-HBRCG. Activity in inhibiting MCF-7cell growth has been shown to be predictive of the potential in vivochemopreventive/chemotherapeutic activity of these analogs.

In summary, both 4-HBRCG and 4-HBRCGlucose inhibit the growth of MCF-7human breast cancer cell in culture, as shown in FIG. 5. 4-HBRCG sharesthe ability with 4-HPR and RA to reduce the size and number of ratmammary tumors. However, a number of the toxic effects shown by theparent retinoids are reduced or eliminated with 4-HBRCG. Thus, thesefully unhydrolyzable analogs have a significant utility and advantage aslow-toxicity chemopreventive/chemotherapeutic agents for use in mammal,including humans.

Pharmaceutical Compositions:

Another aspect of the invention provides pharmaceutical compositions,for medical use, comprising an active compound, i.e., a Formula Icompound or a pharmaceutically-acceptable salt thereof, optionally incombination with an acceptable carrier and optionally in combinationwith other therapeutically-active ingredients or inactive accessoryingredients. The carrier must be pharmaceutically-acceptable in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient. The pharmaceutical compositionsinclude those suitable for oral, topical, inhalation, rectal orparenteral (including subcutaneous, intramuscular and intravenous)administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the pharmaceuticalarts. The term “unit dosage” or “unit dose” is denoted to mean apredetermined amount of the active ingredient sufficient to be effectivefor treating an indicated activity or condition. Making each type ofpharmaceutical composition includes the step of bringing the activecompound into association with a carrier and one or more optionalaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing the active compound into associationwith a liquid or solid carrier and then, if necessary, shaping theproduct into the desired unit dosage form.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,boluses or lozenges, each containing a predetermined amount of theactive compound; as a powder or granules; or in liquid form, e.g., as anoil, aqueous solution, suspension, syrup, elixir, emulsion, dispersion,or the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface-activeor dispersing agents. Molded tablets may be made by molding in asuitable machine a mixture of the powdered active compound with anysuitable carrier.

Formulations suitable for parenteral administration convenientlycomprise a sterile preparation of the active compound in, for example,water for injection, saline, a polyethylene glycol solution and thelike, which is preferably isotonic with the blood of the recipient.

Useful formulations also comprise concentrated solutions or solidscontaining the compound of Formula I which upon dilution with anappropriate solvent give a solution suitable for parenteraladministration.

Preparations for topical or local applications comprise aerosol sprays,lotions, gels, ointments, suppositories etc., andpharmaceutically-acceptable vehicles therefor such as water, saline,lower aliphatic alcohols, polyglycerols such as glycerol, polyethyleneglycerol, esters of fatty acids, oils and fats, silicones, and otherconventional topical carriers. In topical formulations, the subjectcompounds are preferably utilized at a concentration of from about 0.1%to 5.0% by weight.

Compositions suitable for rectal administration, comprise a suppository,preferably bullet-shaped, containing the active ingredient andpharmaceutically-acceptable vehicles therefor such as hard fat,hydrogenated cocoglyceride, polyethylene glycol and the like. Insuppository formulations, the subject compounds are preferably utilizedat concentrations of from about 0.1% to 10% by weight.

Compositions suitable for rectal administration may also comprise arectal enema unit containing the active ingredient andpharmaceutically-acceptable vehicles therefor such as 50% aqueousethanol or an aqueous salt solution which is physiologically compatiblewith the rectum or colon. The rectal enema unit consists of anapplicator tip protected by an inert cover, preferably comprised ofpolyethylene, lubricated with a lubricant such as white petrolatum andpreferably protected by a one-way valve to prevent back-flow of thedispensed formula, and of sufficient length, preferably two inches, tobe inserted into the colon via the anus. In rectal formulations, thesubject compounds are preferably utilized at concentrations of fromabout 0.1 to about 10% by weight.

Useful formulations also comprise concentrated solutions or solidscontaining the active ingredient which upon dilution with an appropriatesolvent, preferably saline, give a solution suitable for rectaladministration. The rectal compositions include aqueous and non-aqueousformulations which may contain conventional adjuvants such as buffers,bacteriostats, sugars, thickening agents and the like. The compositionsmay be presented in rectal single dose or multi-dose containers, forexample, rectal enema units.

Preparations for topical or local surgical applications for treating awound comprise dressings suitable for wound care. In both topical orlocal surgical applications, the sterile preparations of compounds ofFormula I are preferably utilized at concentrations of from about 0.1%to 5.0% by weight applied to a dressing.

Compositions suitable for administration by inhalation includeformulations wherein the active ingredient is a solid or liquid admixedin a micronized powder having a particle size in the range of about 5microns or less to about 500 microns or liquid formulations in asuitable diluent. These formulations are designed for rapid inhalationthrough the oral passage from conventional delivery systems such asinhalers, metered-dose inhalers, nebulizers, and the like. Suitableliquid nasal compositions include conventional nasal sprays, nasal dropsand the like, of aqueous solutions of the active ingredient(s).

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more optional accessoryingredient(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, colorants, binders, surface-activeagents, thickeners, lubricants, suspending agents, preservatives(including antioxidants) and the like.

The amount of compound of Formula I required to be effective for anyindicated condition will, of course, vary with the individual mammalbeing treated and is ultimately at the discretion of the medical orveterinary practitioner. The factors to be considered include thecondition being treated, the route of administration, the nature of theformulation, the mammal's body weight, surface area, age and generalcondition, and the particular compound to be administered. In general, asuitable effective dose is in the range of about 0.05 to about 200 mg/kgbody weight per day, preferably in the range of about 1 to about 70mg/kg per day, calculated as the non-salt form of Formula I. The totaldaily dose may be given as a single dose, multiple doses, e.g., two tosix times per day, or by intravenous infusion for a selected duration.Dosages above or below the range cited above are within the scope of thepresent invention and may be administered to the individual patient ifdesired and necessary.

In general, the pharmaceutical compositions of this invention containfrom about 0.5 mg to about 1.5 g active ingredient per unit dose and,preferably, from about 7.5 to about 1000 mg per unit dose. If discretemultiple doses are indicated, treatment might typically be 100 mg of acompound of Formula I given from two to four times per day.

The compounds according to the present invention may be administeredprophylactically, chronically, or acutely. For example, such compoundsmay be administered prophylactically to inhibit the formation of cancersin the subject being treated. The subject compounds may also beadministered prophylactically to patients suffering a primary cancer toprevent the occurrence of metastatic cancers. In addition to theprevention of primary and metastatic cancers, chronic administration ofthe subject compounds will typically be indicated in treating recurringcancers. Acute administration of the subject compounds is indicated totreat, for example, aggressive cancers prior to surgical or radiologicalintervention.

EXAMPLES

The following Examples are included solely to provide a more completedescription of the invention disclosed and claimed herein. The Examplesdo not limit the scope of the invention in any fashion.

Synthesis:

General methods: Anhydrous THF and CH₂Cl₂ were obtained usingdistillation from sodium benzophenone ketyl and calcium hydride,respectively. Sigma-Aldrich (Milwaukee, Wis.) supplied startingmaterials and reagents. Cambridge Isotopes Laboratories (Cambridge,Mass.) supplied isotope labeled reagents. All reactions and handling ofretinoid-containing compounds were done under gold fluorescent lights.Thin-layer chromatography was performed on Merck (Gibbstown, N.J.)silica gel 60 F₂₅₄ aluminum plates. Column chromatography was performedwith Merck silica gel 60 and reverse phase flash chromatography withMerck Lichroprep® RP-18. Analytical HPLC was done on Beckman Instruments(San Ramon, Calif.), with pump component 127 and detector module 166,equipped with a Metachem Polaris (Varian), 5 μm C-18, 250×4.6 mm column.All retinoids were detected at a wavelength of 350 nm. Melting pointswere determined using a Thomas-Hoover (Philadelphia, Pa.) capillaryapparatus and are uncorrected. Optical rotations were conducted on aPerkin-Elmer (Wellesley, Mass.) 241 polarimeter and reported inmol·dm⁻¹·gram⁻¹. Ultraviolet spectra were recorded on a BeckmanInstruments DU-40 spectrophotometer. Infrared spectra were recorded asfilms on silver chloride plates using a Nicolet (Madison, Wis.) Protégé460 spectrophotometer. NMR spectra were recorded on a Bruker (Billerica,Mass.) DRX 400 spectrometer. Mass spectra were recorded on a Micromass(Milford, Mass.) QTOF Electrospray mass spectrometer.

2,3,4,6-tetra-ο-(methoxymethyl)-D-gluconic acid-δ-lactone (9). To aflame-dried flask under argon atmosphere was added δ-gluconolactone (8)(7.38 g, 41.4 mmol) and CH₂Cl₂ (400 mL). Upon cooling the suspensionwith an ice bath, diisopropylethylamine (57.6 mL, 331 mmol) was addeddropwise, followed by careful addition of chloromethyl methyl ether (50g, 621 mmol) via an addition funnel. A significant amount of white smokeformed in the reaction vessel. Solid tetrabutylammonium iodide (50 g,134 mmol) was added and the solution was allowed to warm to roomtemperature (hereinafter “rt”). The reaction stirred in the dark for 48h upon which the solution gradually turned red. After cooling the vesselto 0° C., saturated aqueous NH₄Cl (75 mL) was added. The contents werethen diluted with water and the layers separated. The organic layer waswashed with brine and the combined aqueous layers were extracted withCH₂Cl₂ (3×). The combined organic layers were dried (MgSO₄), filtered,and concentrated. The solids were then triturated with ether (4×) andthe ether was concentrated. The resultant oil was chromatographed onsilica gel (1:1 hexanes/ethyl acetate) to afford 12.04 g (83%) of clearoil. [α]_(D) 118.4 (c 2.15, CH₂Cl₂); IR (cm⁻¹) 2948 (s), 2885 (s), 1757(s), 1464 (m), 1443 (m), 1213 (s), 1150 (s), 1035 (s), 912 (m); ¹H NMR(CDCl₃) δ 3.36-3.42 (m, 12H), 3.77 (dd, 1H, J=3.8, 11.3 Hz), 3.82 (dd,1H, J=2.8, 11.3 Hz), 3.99-4.05 (m, 2H), 4.29 (d, 1H, J=6.6 Hz),4.55-4.56 (m, 1H), 4.65 (s, 2H), 4.69-4.92 (m, 7H); ¹³C NMR (CDCl₃) δ55.42, 56.05, 56.11, 56.22, 66.12, 73.69, 74.77, 78.43, 96.56, 96.66,96.78, 96.91, 97.13, 168.70; HRMS (ES) calcd for C₁₄H₂₆O₁₀ (M+Na)377.1424. found 377.1408.

Dimethyl titanocene, Cp₂Ti(CH₃)₂ (Petasis' reagent). To a flame-driedflask under argon atmosphere was added titanocene dichloride (14.63 g,58.8 mmol) and absolute ether (300 mL), which was cooled to 10° C.Methyl lithium (100 mL, 140 mmol, 1.4 M) was carefully added dropwisevia an addition funnel in the dark. The cold bath was removed and thered solution was allowed to stir for 10 min. The solution was thencooled to 0° C. and ice water (25 mL) was carefully added to quench theunreacted methyl lithium. The layers were separated and the aqueouslayer extracted with ether (2×). The combined organic layers were dried(Na₂SO₄) under argon for 1 h and concentrated in the dark at 20° C. togive 12.4 g of orange solid. Dry toluene (100 mL) was added and thereagent was stored at 4° C. and used without characterization.

2,6-Anhydro-1-deoxy-3,4,5,7-tetra-ο-(methoxymethyl)-D-gluco-hept-1-enitol(10). To a flame-dried flask under argon atmosphere was added the sugarlactone 9 (10.05 g, 28.4 mmol) dissolved in dry toluene (140 mL) via anaddition funnel. The toluene solution of dimethyl titanocene (12.4 g, 59mmol) was then added dropwise via an addition funnel to give a redsolution. The flask was then equipped with a reflux condenser and heatedto 70° C. and let stir in the dark for 18 h. The resultant blacksolution was cooled and poured into hexanes (˜500 mL). A precipitateformed and was filtered through celite. The supernatant was concentratedto yield a red oil which was chromatographed on silica gel (4:1 then 2:1hexanes/ethyl acetate) to afford 8.66 g (87%) of yellowish oil. [α]_(D)46.8 (c 2.33, CH₂Cl₂); IR (cm⁻¹) 2940 (m), 2895 (m), 1750 (w), 1440 (w),1154 (s), 1032 (s), 918 (m); ¹H NMR (DMK-d₆) δ 3.31-3.37 (m, 12H),3.64-3.71 (m, 2H), 3.78-3.83 (m, 2H), 3.88-3.89 (m, 1H), 4.12 (d, 1H,J=5.4 Hz), 4.35 (s, 1H), 4.51 (s, 1H), 4.62 (s, 2H), 4.66-4.84 (m, 6H);¹³C NMR (DMK-d₆) δ 55.15, 55.87, 56.04, 56.19, 67.50, 75.42, 76.68,77.36, 81.08, 93.43, 95.35, 97.23, 97.64, 97.81, 156.39; HRMS (ES) calcdfor C₁₅H₂₈O₉ (M+Na) 375.1631. found 375.1628.

2,6-Anhydro-1-deoxy-1-[4-(hydroxymethyl)phenyl]-3,4,5,7-tetra-ο-(methoxymethyl)-D-glycero-D-gulo-heptitol(11). To a flame-dried flask under argon atmosphere was added theexocyclic olefin 10 (3.75 g, 10.6 mmol) dissolved in dry THF (100 mL).9-BBN-H (53.2 mL, 26.6 mmol, 0.5 M) was added via addition funnel. Theflask was then equipped with a reflux condenser, heated to 75-80° C.,and refluxed for 4.5 h. The mixture was cooled to rt, then K₃PO₄ (10 mL,3 M) was added and allowed to stir for 10 min. p-Bromobenzyl alcohol(3.98 g, 21.3 mmol) and PdCl₂(dppf) (0.686 g, 0.85 mmol) dissolved inDMF (100 mL) was added via addition funnel and stirred for 18 h. Thereaction was diluted with water and ether, and then the layers wereseparated. The organic layer was washed with water and brine. Thecombined aqueous layers were extracted with ether (3×). The organiclayers were combined, dried (MgSO₄), concentrated, and chromatographed(1:1 then 1:2 hexanes/ethyl acetate) to afford 3.29 g (67%) of orangeoil. [α]_(D) −26.2 (c 1.15, DMK); IR (cm⁻¹) 3470 (w), 2932 (m), 2887(m), 1692 (m), 1444 (w), 1150 (s), 1101 (s), 1024 (s), 918 (m); ¹H NMR(DMK-d₆) δ 2.60 (dd, 1H, J=9.4, 14.4 Hz), 3.18-3.42 (m, 5H), 3.25 (s,3H), 3.35 (s, 3H), 3.40 (s, 3H), 3.44 (s, 3H), 3.54-3.61 (m, 2H), 3.73(dd, 1H, J=1.8, 11.3 Hz), 4.51-4.58 (m, 4H), 4.70 (d, 1H, J=6.5 Hz),4.77-4.85 (m, 4H), 4.93 (d, 1H, J=6.5 Hz), 7.25 (s, 4H); ¹³C NMR(DMK-d₆) δ 38.35, 55.04, 56.45, 56.55, 64.44, 64.57, 67.42, 77.97,79.07, 80.32, 81.63, 84.83, 97.20, 99.01, 99.19, 99.32, 127.15, 130.11,138.75, 141.03; HRMS (ES) calcd for C₂₂H₃₆O₁₀ (M+Na) 483.2206. found483.2188.

2,6-Anhydro-1-deoxy-1-[4-(methoxymethyl)phenyl]-3,4,5,7-tetra-O-(methoxymethyl)-D-glycero-D-gulo-heptitol(12). To a flame-dried flask under argon atmosphere was added theC-glycoside benzyl alcohol 11 (2.44 g, 5.3 mmol) dissolved in dry THF(100 mL). Sodium hydride (0.63 g, 26.5 mmol) was added to the flask andthe suspension stirred for 1.5 h. lodomethane (4.5 g, 31.7 mmol)dissolved in THF (10 mL) was cannulated into the reaction mixture andallowed to stir for 18 h. After cooling in an ice bath, water was addedcarefully to quench excess NaH. The mixture was extracted with ether(3×), the organic layers combined, washed with brine, dried (MgSO₄),concentrated, and then chromatographed (1:1 then 1:2 hexanes/ethylacetate) to give 2.37 g (94%) of clear oil. [α]_(D) −27.0 (c 4.70, DMK);IR (cm⁻¹) 2981 (s), 2883 (s), 1701 (w), 1513 (m), 1444 (m), 1378 (m),1301 (m), 1158 (s), 1105 (s), 1028 (s), 918 (s); ¹H NMR (DMK-d₆) δ2.61(dd, 1H, J=9.4, 14.4 Hz), 3.19-3.42 (m, 5H), 3.24 (s, 3H), 3.30 (s, 3H),3.35 (s, 3H), 3.40 (s, 3H), 3.44 (s, 3H), 3.54-3.64 (m, 2H), 3.73 (dd,1H, J=2.6, 13.5 Hz), 4.38 (s, 2H), 4.50 (d, 1H, J=6.4 Hz), 4.54 (d, 1H,J=6.4 Hz), 4.70 (d, 1H, J=6.5 Hz), 4.77-4.85 (m, 4H), 4.93 (d, 1H, J=6.5Hz), 7.21 (d, 2H, J=8.0 Hz), 7.28 (d, 2H, J=8.0 Hz); ¹³C NMR (DMK-d₆)δ33.39, 55.05, 56.47, 56.49, 56.57, 57.97, 67.46, 74.84, 78.00, 79.10,80.23, 81.66, 84.86, 97.20, 99.01, 99.21, 99.32, 128.15, 130.19, 137.23,139.45; HRMS (ES) calcd for C₂₃H₃₈O₁₀ (M+Na) 497.2363. found 497.2384.

2,6-Anhydro-7-deoxy-7-[4-(methoxymethyl)phenyl]-3,4,5-tri-ο-acetyl-L-glycero-L-gulo-heptinoicacid methyl ester (13). The MOM-protected glucoside 12 (2.43 g, 5.12mmol) dissolved in methanol (500 mL) was placed in a flask at rt.Aqueous HCl (6 N, 26 mL) was added and the solution stirred for 18 hafter which the mixture was then concentrated to dryness and set aside.In a separate flask, KBr (2.42 g, 20.38 mmol) and TEMPO (3.19 g, 20.41mmol) were added to a saturated NaHCO₃ solution (400 mL) and stirred for20 min at 0° C. Aqueous NaOCl (11.2 mL, 1.6-2.0 M) was then added andstirred for 10 min. The deprotected sugar was dissolved in saturatedNaHCO₃ solution (100 mL) and added to the flask with the TEMPO mixture.The total mixture was stirred for 45 min at 0° C. Then the reaction wasquenched with EtOH (50 mL) and the contents were washed with ether in aseparatory funnel. The aqueous layer was concentrated to dryness and theremaining solid was exhaustively triturated with methanol. The methanolwas then concentrated and dried. The dried residue was suspended in DMF(180 mL) and then iodomethane (6.4 g) dissolved in DMF (10 mL) was addedand allowed to stir for 20 h under argon at rt. The reaction mixture wasthen supplemented with acetic anhydride (40 mL), pyridine (20 mL), andDMAP (15 mg) and allowed to stir for 18 h. The reaction mixture wasdiluted with water and extracted (3×) with ethyl acetate. The organiclayers were washed with water, brine, dried (MgSO₄), concentrated, andchromatographed (2:1 then 1:1 hexanes/ethyl acetate) to give 1.90 g(82%) of clear oil that solidified upon standing, mp 84-86° C. [α]_(D)−13.04 (c 1.15, DMK); IR (cm⁻¹) 2956 (w), 2818 (w), 1750 (s), 1440 (m),1370 (m), 1211 (s), 1105 (m), 1028 (m); ¹H NMR (DMK-d₆) δ 1.94 (s, 3H),1.94 (s, 3H), 1.95 (s, 3H), 2.74-2.81 (m, 1H), 2.90 (dd, 1H, J=3.4, 7.3Hz), 3.30 (s, 3H), 3.65 (s, 3H), 3.94-3.99 (m, 1H), 4.18 (d, 1H, J=9.8Hz), 4.38 (S, 2H), 4.90 (t, 1H, J=9.8 Hz), 5.05 (t, 1H, J=9.8 Hz), 5.29(t, 1H, J=9.8 Hz), 7.22 (s, 4H); ¹³C NMR (DMK-d₆) δ 20.39, 20.52, 20.60,38.12, 52.67, 58.03, 70.62, 72.53, 74.09, 74.73, 76.41, 78.62, 128.25,130.16, 137.43, 137.76, 168.40, 169.89, 170.07, 170.30; HRMS (ES) calcdfor C₂₂H₂₈O₁₀ (M+Na) 475.1580. found 475.1577.

2,6-Anhydro-7-deoxy-7-[4-(bromomethyl)phenyl]-3,4,5-tri-ο-acetyl-L-glycero-L-gulo-heptinoicacid methyl ester (14). To a dry flask equipped with a CaSO₄ drying tubewas added the C-glucuronide methyl ether 13 (462 mg, 1.02 mmol) alongwith 30% HBr in acetic acid (5 mL, 25 mmol) at 0° C. The mixture stirredfor 30 min and then at rt for 18 h. The mixture was diluted withmethylene chloride and then carefully washed with water and saturatedNaHCO₃ solution. The organic layer was dried (MgSO₄), concentrated, andchromatographed (2:1 then 1:1 hexanes/ethyl acetate) to give 440 mg(86%) of white foam, which was crystallized with ether, mp 116-117° C.[α]_(D) −12.03 (c 5.57, DMK); IR (cm⁻¹) 3026 (w), 2952 (w), 1754 (s),1440 (m), 1370 (m), 1215 (s), 1101 (m), 1036 (m); ¹H NMR (DMK-d₆) δ 1.93(s, 3H), 1.94 (s, 3H), 1.95 (s, 3H), 2.76-2.83 (m, 1H), 2.92 (dd, 1H,J=3.5, 7.3 Hz), 3.64 (s, 3H), 3.96-3.99 (m, 1H), 4.20 (d, 1H, J=9.7 Hz),4.62 (s, 2H), 4.90 (t, 1H, J=9.7 Hz), 5.05 (t, 1H, J=9.7 Hz), 5.29 (t,1H, J=9.7 Hz), 7.25 (d, 2H, J=8.2 Hz), 7.36 (d, 2H, J=8.2 Hz); ¹³C NMR(DMK-d₆) δ 20.40, 20.52, 20.63, 34.37, 38.12, 52.69, 70.58, 72.52,74.04, 76.35, 78.43, 129.88, 130.68, 137.26, 138.67, 168.39, 169.91,170.09, 170.29; HRMS (ES) calcd for C₂₁H₂₅BrO₉ (M+Na) 523.0580. found523.0602.

tert-Butyl-dimethylsilylcyanohydrin of retinal (16). To a flame-driedflask under argon atmosphere was added retinal (15) (1.03 g, 3.62 mmol)dissolved in dry CH₂Cl₂ (50 mL). A catalytic amount of Et₃N (0.1 mL) wasadded then tert-butyldimethylsilyl cyanide (1.0 g, 7.08 mmol) dissolvedin CH₂Cl₂ (10 mL) was added by cannulation. The reaction stirred for 20h after which the solution was concentrated, chromatographed (95:5hexanes/ethyl acetate), dried (Na₂SO₄) under argon, and subjected tovacuum overnight to give 1.20 g (78%) of orange oil. UV λ_(max)=329 nm(ε=49462); IR (cm⁻¹) 3042 (w), 2960 (s), 2928 (s), 2850 (s), 2239 (w),1586 (w), 1472 (m), 1358 (m), 1256 (m), 1105 (s), 963 (s), 832 (s), 775(m); ¹H NMR (DMK-d₆) δ 0.16 (s, 3H), 0.20 (s, 3H), 0.90 (s, 9H), 1.02(s, 6H), 1.45-1.48 (m, 2H), 1.58-1.63 (m, 2H), 1.70 (s, 3H), 1.99 (s,6H), 5.57-5.61 (m, 2H), 6.13-6.23 (m, 3H), 6.38 (d, 1H, J=15.2 Hz), 6.86(dd, 1H, J=11.3, 15.2 Hz); HRMS (ES) calcd for C₂₇H₄₃NOSi (M+Na)448.3012. found 448.2982.

2,6-Anhydro-7-deoxy-7-[4-(retinoylmethyl)-phenyl]-3,4,5-tri-ο-acetyl-L-glycero-L-gulo-heptinoicacid methyl ester (18). To a flame-dried flask under argon atmospherewas added THF (40 mL) along with LiHMDS (1.0 M in hexanes, 3.8 mL, 3.8mmol). The mixture was cooled to −78° C. upon which the silylcyanohydrin of retinal 16 (1.08 g, 2.54 mmol) in THF (15 mL) was addedby cannulation into the flask. The dark red solution was allowed to stirfor 30 min at −78° C. The crystalline bromoglucuronide 14 (2.78 g, 5.56mmol) in THF (15 mL) was cannulated into the flask and the mixturestirred for 3 h at −78° C. after which the solution changed to lightred. The reaction was taken out of the cold bath and quenched with asolution of 1 M NH₄Cl (10 mL). The mixture was extracted with ethylacetate (3×) and the organic layers were combined, washed with brine,dried (Na₂SO₄), filtered, concentrated, and chromatographed (2:1hexanes/ethyl acetate) to give 1.0 g (47%) of yellow foam 17 and 1.7 gof recovered bromide 14. The alkylated product was taken up in 1%aqueous THF (200 mL) and chilled to 0° C. TBAF (309 mg, 1.18 mmol) wasadded and the darkened solution stirred 1 h. The reaction was dilutedwith water and extracted with ethyl acetate (3×). The organic layerswere combined, washed with brine, dried (NaSO₄), filtered, concentrated,and chromatographed (2:1 hexanes/ethyl acetate) to give 628 mg (35% overtwo steps) of yellow foam. UV λ_(max)=379 nm (ε=36182); HPLC t_(R)=24.0min, 1 mL/min (85:15 MeOH:H₂O both with 10 mM NH₄OAc); IR (cm⁻¹) 2956(w), 2924 (w), 2863 (w), 1754 (s), 1672 (w), 1554 (m), 1436 (w), 1362(w), 1215 (s), 1081 (w), 1028 (w), 971 (w); ¹H NMR (DMK-d₆) δ 1.02 (s,6H), 1.45-1.48 (m, 2H), 1.58-1.62 (m, 2H), 1.69 (s, 3H), 1.90 (s, 3H),1.93 (s, 3H), 1.95 (s, 3H), 2.01 (s, 3H), 2.03-2.05 (m, 2H), 2.28, (s,3H), 2.75-2.89 (m, 2H), 3.64 (s, 3H), 3.71 (s, 2H), 3.95-3.98 (m, 1H),4.19 (d, 1H, J=9.8 Hz), 4.90 (t, 1H, J=9.8 Hz), 5.05 (t, 1H, J=9.8 Hz),5.29 (t, 1H, J=9.8 Hz), 6.15-6.35 (m, 5H), 7.13-7.20 (m, 5H); ¹³C NMR(DMK-d₆) δ 13.45, 14.68, 20.41, 20.96, 21.08, 21.15, 22.47, 34.15,35.41, 38.76, 40.86, 52.25, 53.23, 71.18, 73.09, 74.63, 76.93, 79.13,126.89, 129.86, 130.73, 130.93, 131.01, 131.47, 133.69, 134.89, 135.11,137.14, 137.26, 138.95, 139.09, 140.96, 152.68, 168.97, 170.45, 170.64,170.84, 198.78; HRMS (ES) calcd for C₄₁H₅₂O₁₀ (M+Na) 727.3458. found727.3456.

2,6-Anhydro-7-deoxy-7-[4-(retinoylmethyl)-phenyl]-L-glycero-L-gulo-heptinoicacid (7). To a flask was added the protected glucuronide-retinoidconjugate 18 (1.15 g, 1.64 mmol) dissolved in methanol (500 mL) andchilled to 4° C. Potassium carbonate (136 mg, 0.98 mmol) was added andallowed to stir for 20 h. The reaction mixture was concentrated at25-30° C. to ˜200 mL. Adjustment to the original volume with methanolwas followed by addition of 1 N KOH (14 ml, 14 mmol). After stirring for20 h at 4° C., the reaction was warmed and allowed to stir for 5 h atrt. The reaction was then cooled to 0° C. and carefully adjusted to pH 7with 4 N HCl. The reaction mixture was concentrated at 25-30° C. to ˜100mL, cooled back to 0° C., and the pH carefully adjusted to 3 with 1 NHCl. The suspension was extracted with ethyl acetate and the organiclayers were combined, dried (Na₂SO₄) under argon for 2 h, and carefullyconcentrated. The residue was chromatographed on reverse phase silicagel (gradient 70:30 to 85:15 methanol/water) to yield 759 mg (82%) ofyellow foam, which was stored at −80° C. until needed. UV λ_(max)=382 nm(ε=30019); HPLC t_(R)=9.2 min (1 mL/min, 85:15 MeOH:H₂O both with 10 mMNH₄OAc); IR (cm⁻¹) 3384 (br), 2920 (s), 1721 (m), 1664 (s), 1550 (s),1427 (m), 1362 (m), 1232 (w), 1089 (m), 1052 (m), 1102 (s), 967 (w); ¹HNMR (MeOH-d₄) δ 0.94 (s, 6H), 1.38-1.41 (m, 2H), 1.54-1.58 (m, 2H), 1.61(s, 3H), 1.91 (s, 3H), 1.93-1.96 (m, 2H), 2.20 (s, 3H), 2.60 (dd, 1H,J=8.7, 14.4 Hz), 3.03-3.08 (m, 2H), 3.21-3.29 (m, 2H), 3.37 (t, 1H,J=9.5 Hz), 3.53 (d, 1H, J=9.5 Hz), 3.62 (s, 2H), 6.03-6.25 (m, 5H),7.02-7.16 (m, 5H); ¹³C NMR (MeOH-d₄) δ 12.88, 14.45, 20.29, 21.04,21.93, 29.40, 34.00, 35.25, 38.34, 40.76, 52.11, 73.44, 74.63, 79.28,80.36, 82.36, 124.37, 126.26, 129.92, 130.19, 130.86, 131.01, 131.12,134.00, 134.23, 136.82, 138.70, 138.91, 139.04, 141.12, 154.22, 173.26,201.21; HRMS (ES) calcd for C₃₄H₄₄O₇ (M+Na) 587.2985. found 587.2989.

2,6-Anhydro-1-deoxy-1-[4(methoxymet6hyl)phenyl]-3,4,5,7-tetra-ο-acetyl-D-glycero-D-gulo-heptitol(20). The MOM-protected glucoside 12 (0.643 g, 1.35 mmol) dissolved inmethanol (34 mL) was placed in a flask at rt. Aqueous HCl (6 N, 6.7 mL)was added and the solution stirred for 18 h. The mixture was thenconcentrated to dryness. Acetic anhydride (4 mL) and pyiridine (3 mL)were added to the paste, along with a catalytic amount of DMAP and themixture was allowed to stir for 18 h at rt. The reaction was dilutedwith water and extracted with ethyl acetate (3×). The organic layerswere combined, washed with water and brine, dried (MgSO₄), filtered,concentrated, and chromatographed (1:1 hexanes/ethyl acetate) to give570 mg (90%) of white solid, mp 120-122° C. [α]_(D) −4.0 (c 0.78, DMK);IR (cm⁻¹) 2940 (w), 2862 (w), 1750 (s), 1436 (w), 1370 (m), 1224 (s),1105 (m), 1032 (m); ¹H NMR (CDCl₃) δ 1.96-2.02 (m, 12H), 2.78 (s, 2H,J=5.8 Hz), 3.36 (s, 3H), 3.52-3.57 (m, 2H), 4.02 (dd, 1H, J=2.3, 12.2Hz), 4.20 (dd, 1H, J=5.3, 12.2 Hz), 4.40 (s, 2H), 4.92 (t, 1H, J=9.6Hz), 5.03 (t, 1H, J=9.6 Hz), 5.15 (t, 1H, J=9.6 Hz), 7.16 (d, 2H, J=8.0Hz), 7.23 (d, 2H, J=8.0 Hz); ¹³C NMR (DMK-d₆) δ 20.55, 20.57, 20.65,38.08, 58.02, 63.05, 69.70, 72.81, 74.76, 74.86, 76.05, 78.60, 128.20,130.21, 137.65, 137.75, 170.04, 170.16, 170.37, 170.59; HRMS (ES) calcdfor C₂₃H₃₀O₁₀(M+Na) 489.1737. found 489.1727.

2,6-Anhydro-1-deoxy-1-[4-(bromomethyl)phenyl]-3,4,5,7-tetra-ο-acetyl-D-glycero-D-gulo-heptitol(21). To a dry flask equipped with a drying tube was added theC-glycoside methyl ether 20 (0.54 g, 1.16 mmol) along with 30% HBr inacetic acid (5 mL, 25 mmol) at 0° C. The mixture was stirred for 30 minand then left at rt for 18 h. The mixture was diluted with methylenechloride and then carefully washed with water and saturated NaHCO₃solution. The organic layer was dried (MgSO₄), concentrated, andchromatographed (2:1 then 1:1 hexanes/ethyl acetate) to give 593 mg(97%) of white solid, mp 141-142° C. [α]_(D) −4.67 (c 2.57, DMK); IR(cm⁻¹) 2993 (w), 2952 (w), 1754 (s), 1440 (w), 1374 (m), 1244 (s), 1105(w), 1052 (m); ¹H NMR (DMK-d₆) δ 1.92-1.98 (m, 12H), 2.72 (dd, 1H,J=7.3, 8.6 Hz), 2.88 (dd, 1H, J=3.2, 7.3 Hz), 3.77-3.85 (m, 2H), 4.00(dd, 1H, J=2.4, 6.1 Hz), 4.21 (dd, 1H, J=5.9, 6.1 Hz), 4.63 (s, 2H),4.86 (t, 1H, J=2.6 Hz), 4.97 (t, 1H, J=9.6 Hz), 5.22 (t, 1H, J=9.6 Hz),7.25 (d, 2H, J=8.2 Hz); 7.37 (d, 2H, J=8.2 Hz); ¹³C NMR (DMK-d₆) δ20.49, 20.60, 34.32, 38.07, 63.03, 69.23, 72.82, 74.85, 76.07, 78.40,129.72, 130.68, 137.12, 138.92, 169.97, 170.10, 170.30, 170.52; HRMS(ES) calcd for C₂₂H₂₇BrO₉ (M+Na) 537.0736. found 537.0724.

2,6-Anhydro-7-deoxy-7-[4-(retinoylmethyl)-phenyl]-3,4,5,7-tetra-ο-acetyl-D-glycero-D-gulo-heptinol(22). To a flame dried flask under argon atmosphere was added THF (10mL) along with LiHMDS (1.0 M in hexanes, 0.78 mL, 0.78 mmol) The mixturewas cooled to −78° C. upon which the silyl cyanohydrin of retinal 16(218 mg, 51 mmol) in THF (5 mL) was added by cannulation into the flask.The dark red solution was allowed to stir for 30 min at −78° C. Thecrystalline glucoside bromide 21 (277 mg, 0.53 mmol) in THF (5 mL) wascannulated into the flask and the mixture stirred for 2 h at −78° C.,after which the solution changed to light red. The reaction was takenout of the cold bath and quenched with a solution of 1 M NH₄Cl (1 mL).The mixture was extracted with ethyl acetate (3×) and the organic layerswere combined, washed with brine, dried (Na₂SO₄), filtered, andconcentrated. The crude alkylated product was taken up in 1% aqueous THF(20 mL) and chilled to 0° C. TBAF (134 mg, 0.51 mmol) was added and thedarkened solution stirred overnight while warming to rt. The reactionwas diluted with water and extracted with ethyl acetate (3×). Theorganic layers were combined, washed with brine, dried (Na₂SO₄),filtered, concentrated, and chromatographed (2:1 hexanes/ethyl acetate)to give 132 mg (36% over two steps) of yellow foam. ¹H NMR (DMK-d₆) δ1.00 (s, 6H), 1.43-1.46 (m, 2H), 1.58-1.59 (m, 2H), 1.68 (s, 3H), 1.90(s, 3H), 1.92 (s, 3H), 1.94 (s, 3H), 1.95 (s, 3H), 2.26, (s, 3H),2.70-2.87 (m, 2H), 3.30 (s, 2H), 3.74-3.83 (m, 2H), 3.97 (d, 1H, J=12.0Hz), 4.19 (dd, 1H, J=5.9, 12.0 Hz) 4.85 (t, 1H, J=9.6 Hz), 4.95 (t, 1H,J=9.6 Hz), 5.20 (t, 1H, J=9.6 Hz), 6.15-6.37 (m, 5H), 7.10-7.17 (m, 5H);HRMS (ES) calcd. for C₄₂H₅₄O₁₀(M+Na) 741.3615. found 741.3617.

2,6-Anhydro-7-deoxy-7-[4-(retinoylmethyl)-phenyl]-D-glycero-D-gulo-heptinol(23). To a flask was added the protected glucoside-retinoid conjugate 22(130 mg, 0.18 mmol) dissolved in methanol (75 mL) and chilled to 4° C.Potassium carbonate (25 mg, 0.18 mmol) was added and allowed to stir for20 h. The reaction was then cooled to 0° C. and carefully adjusted to pH5 with 1 N HCl. The solution was extracted with ethyl acetate and theorganic layers were combined, dried (Na₂SO₄), and carefullyconcentrated. The residue was chromatographed on reverse-to phase silicagel (gradient 70:30 to 85:15 methanol/water) to yield 49 mg (49%) ofyellow foam. UV λmax=380 nm (ε=34567); HPLC t_(R)=13.8 min (1 mL/min,85:15 MeOH:H₂O both with 10 mM NH₄OAc); IR (cm⁻¹) 3388 (br), 2948 (m),2846 (m), 1652 (m), 1554 (w), 1456 (w), 1415 (w), 1113 (w), 1056 (m),1016 (s), 694 (br); ¹H NMR(DMK-d₆) δ 1.02 (s, 6H), 1.45-1.48 (m, 2H),1.58-1.64 (m, 2H), 1.69 (s), 3H), 2.01 (s, 3H), 2.03-2.05 (m, 2H), 2.29,(s, 3H), 2.65 dd, 1H, J=8.3, 14.3 Hz), 3.09-3.38 (m, 6H), 3.57-3.60 (m,1H), 3.70 (s, 3H), 6.15-6.35 (m, 5H), 7.12-7.26 (m, 5H); ¹³C NMR(DMK-d₆) δ 13.43, 14.66, 20.41, 22.46, 26.31, 30.95, 34.14, 38.69,40.85, 52.34, 63.81, 72.70, 75.05, 80.37, 81.31, 81.50, 129.82, 130.43,130.90, 131.20, 131.48, 133.64, 134.46, 137.31, 138.96, 139.08, 140.89,152.57, 198 97; HRMS (ES) calcd for C₃₄H₄₆O₆ (M+Na) 573.3192. found573.3204.

Biological:

Animal Studies: Mammary tumors were induced by intragastric intubationof 50-day old female Sprague-Dawley rats (Harlan Industries,Indianapolis, Ind.) with a single dose of 15 mg DMBA in 1.0 ml of sesameoil per rat. The rats were then maintained on a powdered Teklad 22/5rodent chow diet (W) 8640 (Harlan Industries, Indianapolis, Ind.), andallowed food and water ad libitum. Four months later, rats which haddeveloped palpable tumors were randomly assigned to the experimentalgroups (4 rats/group). The retinoid-treated groups were fed dietssupplemented with 2 mmol/kg diet of atRA, 4-HPR, or 4-HBRCG,respectively. The retinoids were added to the diet in a vehicleconsisting of 25 ml of ethanol: tricaprylin (1:4 v/v) plus 2% (w/v) ofα-tocopherol as previously described.⁹ This vehicle was also added tothe control diet. The additives were blended into the chow diets using aHobart food mixer. The diets were fed in stainless steel feedersdesigned with food hoppers. The food was replaced weekly with freshlyprepared diets. Food consumption was determined once weekly, and fromthat the average daily consumption/rat was estimated. These diets werecontinuously fed for 22 days. Animals were also weighted weekly andmonitored for general health status and signs of possible toxicity dueto treatment.

Baseline measurement of initial tumor sizes, numbers and rat bodyweights were determined immediately before commencement of treatments,and final measurements were recorded just prior to sacrifice of theanimals. Animals were palpated for tumors twice weekly and tumordiameters were measured weekly by a micrometer caliper. Tumor volumeswere calculated using the formula [V= 4/3 πr³] where r is one-half themean of the sum of the largest diameter and the axis at right angle toit. All tumors as well as lungs, liver, kidney and femur were excised atthe end of the experiment for chemical and histopathological evaluation.Blood samples were also taken from each animal for determination ofplasma retinol and triglyceride levels.

Plasma Triglyceride Measurement. Bloods were drawn from anesthetizedanimals in the presence of EDTA as an anticoagulant, and the resultingplasma was used for the measurement of plasma “true” triglyceride levelsusing a kit from Sigma-Aldrich (Saint Louis, Mo.). Briefly, the totalplasma triglyceride and glycerol concentrations were determined, and theglycerol component was subtracted from the total plasma triglyceridemeasurement to obtain the “true” serum triglyceride concentration.

Plasma Retinoid Assay. To 500 μL of plasma was added 150 μL of ethanolcontaining 0.75 μg of internal standard (N-(4-chlorophenyl) retinamide).After mixing 30 sec., 500 μL of ethyl acetate was added followed by 1min. of mixing and centrifugation for 5 minutes at 1000 rpm in an IEC CLcentrifuge. The ethyl acetate layer was removed and syringe filteredthrough a 0.45 um filter. The ethyl acetate extraction was repeated twomore times. The combined extracts were evaporated and the residuereconstituted in 100 μL of methanol. The methanol extract (20 μL) wasanalyzed by HPLC on a Beckman Instruments model 127 instrument equippedwith a model 166 UV detector. Chromatography was done on a precolumnequipped 250×4.6 mm Bechman Ultrasphere ODS column with an 85%methanol/water mobile phase (both containing 10 mM ammonium acetate)flowing at 1 mL/min. Analysis for both internal standard and retinol wasconducted at 350 nm and internal standard recoveries and retinol levelswere determined by comparison with standard curves, with adjustment ofthe retinol level based on recovery. Recoveries of internal standardaveraged ca. 78%. Previous extraction of plasma from vitamin A deficientrats showed no substances interfering with the elution position of theretinol or internal standard. In the 4-HPR treated group, plasma levelsof this retinoid were evaluated simultaneously in the same samples asabove. In order to avoid interfering substances, plasma treatmentretinoid levels for RA and 4-HBRCG were measured using the above systemand a step gradient of 75% methanol for 15 min. followed by 85% methanolfor 40 minutes.

Bone Mineral Content. The femur was disarticulated from the leg, and theadhering soft tissue was removed by dissection. Femurs were scannedusing the Lunar PIXImus 2 system (Model X2608, General Electric usingthe LUNAR software version 1.45), and control measurements were madeusing the small animal quality control phantom. Femurs were scanned 5times each with re-positioning at each measure. The average value of thebone mineral content (BMC) in grams for each animal is reported as oneindependent measure.

Nuclear Retinoid Receptor Binding Assay. Competition of 3 and 7 and4-HBRCGlucose with [³H]-all-trans-RA (4.2-4.6 nM) for binding to RARβand RARγ and with [³H]-9-cis RA (1.9 nM) for binding to RXRγ wasdetermined using an in vitro ligand binding assay.^(41, 42)[³H]-all-trans-RA (40.5 Ci/mmole) or [³H]-9-cis-RA (69.4 Ci/mmole) wasadded to receptor-containing extracts in the absence and presence ofincreasing concentrations of competing ligands at 4° C. for 3 hr. Ahydroxylapatite (HAP) assay was used to separate ligand bound toreceptor from that free in solution, and the radioactivity associatedwith the HAP pellet was measured by scintillation counting.

Isolation of RNA and quantitative PCR. Total and polyA⁺ RNA was isolatedas described.⁴³ Briefly, lung and liver tissue was collected andflash-frozen in liquid nitrogen until use. Tissue (0.5 to 1 g) washomogenized in buffer (1:10; wt/vol), and total RNA was isolatedaccording to the method of Chomeczynski and Sacchi.⁴⁴

A rat CYP26A1 partial cDNA was generated by PCR amplification from E10.5day rat embryo cDNA. The upstream (5′ GCA GAT GAA GCG CAG GAA ATA CG 3′)(SEQ. ID. NO: 1) and downstream (5′ CCC ACG AGT GCT CAA TCA GGA 3′)(SEQ. ID. NO: 2) primers were designed based on the murine cDNA(gi:6681100). The 635 bp cDNA was subcloned into pGEM-Teasy (Promega,Madison, Wis.) and sequenced. Similarly, a rat CYP26B1 partial cDNA wasgenerated by PCR amplification from E11.5 day rat embryo cDNA. Theupstream (5′ GCT ACA GGG TTC CGG CTT CCA GTC 3′) (SEQ. ID. NO: 3) anddownstream (5′ TCC AGG GCG TCC GAG TAG TCT TTG 3′) (SEQ. ID. NO: 4)primers were designed based on the murine cDNA (gi:31341987), and the606 bp control cDNA was subcloned and sequenced.

The quantitative polymerase chain reaction (Q-PCR) assay was performedusing the real-time LightCycler system (Roche, Indianapolis, Ind., USA)with LightCycler faststart DNA master SYBR green 1 kit (Roche,Indianapolis Ind.) according to the manufacturer's protocols. Poly(A)⁺RNA (0.5 to 1 μg) was reverse transcribed (RT) using AMV enzyme(Promega, Madison, Wis.) and random hexamers. The following primer setswere used for Q-PCR: CYP26A1, upstream 5′-ATG ATT CCT CGC ACA AGC AG-3′(SEQ. ID. NO: 5), downstream 5′-GCT CCA GAC AAC CGC TCA CT-3′ (SEQ. ID.NO: 6); CYP26B1, upstream 5′-AGG CCC AGC GAC TTA CCT TC-3′ (SEQ. ID. NO:7), downstream 5′-AGG GCG TCC GAG TAG TCT TT-3′ (SEQ. ID. NO: 8); andGAPDH, upstream 5′-TGA AGG TCG GTG TGA ACG GAT TTG GC-3′ (SEQ. ID. NO:9), downstream 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′ (SEQ. ID. NO: 10).The primer sets for CYP26A1, CYP26B1 and GAPDH amplify 409 bp to 519 bp(gi:18426827), 708 bp to 957 bp (gi:31220748), and 854 bp to 1836 bp(gi:31377487), respectively.

Growth inhibition of MCF-7 human breast cancer cells. MCF-7 cells wereobtained from the American Type Culture Collection, Manassas, Va. Theywere maintained in DMEM medium supplemented with 4 g/L glucose, 3.7 g/Lsodium bicarbonate and 10% heat-inactivated fetal calf serum. Cells werepassaged into 12-well plates, cultured for 24 hours, after which timethey were dosed with vehicle (0.2% ethanol) or retinoids; medium waschanged and fresh vehicle or retinoids were added daily. Cells wereharvested and the number of live cells was assessed using fluoresceindiacetate, which yields a fluorescent product upon cleavage bymetabolically active cells. Using fluorescent microscopy, at least 200cells/well were counted with a hemacytometer.

Statistical analysis. Descriptive statistics on tumor volumes, tumornumbers, retinol, triglyceride levels and BMC were examined and comparedamong the experimental groups. The statistical significance of thegroups' comparisons was obtained using analysis of variance (ANOVA),ANOVA with repeated measures, and non-parametric tests. Values wereconsidered significant when the p≦0.05.

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What is claimed is:
 1. A compound of Formula I:

wherein X is CH₂; Y is C₁-C₆ alkylene; and R is selected from the groupconsisting of hydrogen,

wherein R¹ is selected from the group consisting of H, COOH, andC₁-C₆-hydroxyalkyl; R² is selected from the group consisting of H and═O; or a salt thereof.
 2. The compound of claim 1, wherein R ishydrogen.
 3. The compound of claim 1, wherein R is


4. The compound of claim 3, wherein R¹ is H.
 5. The compound of claim 3,wherein R¹ is COOH.
 6. The compound of claim 3, wherein R¹ is CH₂OH. 7.The compound of claim 1, wherein R is


8. The compound of claim 1, wherein R² is H.
 9. The compound of claim 1,wherein R² is ═O.
 10. A pharmaceutical composition for inhibitingformation of breast cancer and treating breast cancer in mammals, thecomposition comprising an effective cancer cell growth-inhibiting amountof a compound according to claim 1, or a pharmaceutically-suitable saltthereof, optionally in combination with a pharmaceutically-suitablecarrier.
 11. The composition of claim 10, comprising the compoundwherein R is hydrogen.
 12. The composition of claim 10, comprising thecompound wherein R is


13. The composition of claim 12, comprising the compound wherein R¹ isH.
 14. The composition of claim 12, comprising the compound wherein R¹is COOH.
 15. The composition of claim 12, comprising the compoundwherein R¹ is CH₂OH.
 16. The composition of claim 10, comprising acompound according to claim 1 wherein R is


17. The composition of claim 10, wherein R² is H.
 18. The composition ofclaim 10, wherein R² is ═O.
 19. A method of inhibiting formation ofbreast cancer and treating breast cancer in mammals, the methodcomprising administering a cancer cell growth-inhibiting amount of acompound according to claim 1, or a pharmaceutically-suitable saltthereof, to a patient in need thereof.
 20. The method of claim 19,wherein a compound according to claim 1 wherein R is hydrogen isadministered to the patient.
 21. The method of claim 19, wherein acompound according to claim 1 wherein R is

is administered to the patient.
 22. The method of claim 21, wherein acompound wherein R¹ is H is administered to the patient.
 23. The methodof claim 21, wherein a compound wherein R¹ is COOH is administered tothe patient.
 24. The method of claim 21, wherein a compound wherein R¹is CH₂OH is administered to the patient.
 25. The method of claim 19,wherein a compound according to claim 1 wherein R is

is administered to the patient.
 26. The method of claim 19, wherein acompound according to claim 1 wherein R² is H is administered to thepatient.
 27. The composition of claim 19, wherein a compound accordingto claim 1 wherein R² is ═O is administered to the patient.
 28. Themethod of claim 19, wherein the compound is administered to a humanpatient in need thereof.